CN109957535B

CN109957535B - Bacillus simplex, microbial agent prepared by using bacillus simplex, biological fertilizer and application

Info

Publication number: CN109957535B
Application number: CN201910378531.7A
Authority: CN
Inventors: 王占武; 彭杰丽; 胡栋; 张翠绵; 贾楠; 马佳
Original assignee: Institute Of Genetics And Physiology Hebei Academy Of Agriculture And Forestry Sciences (hebei Agricultural Product Quality Safety Research Center Of Agriculture And Forestry Academy)
Current assignee: INSTITUTE OF AGRICULTURAL RESOURCES AND ENVIRONMENT HEBEI ACADEMY OF AGRICULTURE AND FORESTRY SCIENCES
Priority date: 2019-05-08
Filing date: 2019-05-08
Publication date: 2020-09-11
Anticipated expiration: 2039-05-08
Also published as: CN109957535A

Abstract

The invention provides a broad-spectrum functional bacterium, namely simple Bacillus (Bacillus simplex) CGMCC No.16458, which has high salt tolerance, multifunctional repair continuous cropping, saline-alkali and other obstacle soil, has high capacity of dissolving phosphorus, fixing nitrogen, producing siderophin, ACCD and cytokinin, has strong antagonistic action on Pythium, Fusarium, Curvularia lunata, microsporum and Phytophthora, is suitable for producing biofertilizer with multiple functions of disease prevention, growth promotion and saline-alkali resistance, is applied to grain production and pasture cultivation in medium and heavy saline-alkali lands, and can promote emergence, promote root development, prevent and control root diseases, improve crop yield and quality equivalent effect, remarkably reduce the use of chemical fertilizers and pesticides and have application prospect.

Description

Bacillus simplex, microbial agent prepared by using bacillus simplex, biological fertilizer and application

Technical Field

The invention relates to a microbial strain and application thereof, in particular to simple bacillus, a microbial agent prepared by using the simple bacillus, a biological fertilizer and application thereof.

Background

With the development of society and the increase of population, the demand for arable land resources is increasing. 9913 million hectares of saline-alkali soil exist in China, wherein 70 million hectares of Bohai sea are important potential cultivated land resources. The saline-alkali soil is divided into mild saline-alkali soil, moderate saline-alkali soil and heavy saline-alkali soil. The pH value of the mild saline-alkali soil is 7.1-8.5, the salt content is less than 0.3%, and the plant emergence rate is generally 70% -80%; the pH value of the moderate saline-alkali soil is 8.5-9.5, and the salt content is 0.3% -0.6%; the pH value of the severe saline-alkali soil is more than 9.5, the salt content is more than or equal to 0.6%, and the emergence rate is lower than 50%. Due to unreasonable cultivation and fertilization, the vegetable field is generally subjected to secondary salinization, and soil degradation occurs. The production performance of saline-alkali soil and degraded soil is low, so that the diseases are aggravated, the yield and the quality are reduced, and the industrial competitiveness and the health of consumers are influenced. The biological improvement method for improving the salt tolerance growth promotion and the yield of the degraded soil crops of the saline-alkali soil crops by utilizing the rhizosphere growth-promoting microorganisms has the characteristics of less investment, quick response and high benefit, and becomes a research hotspot at home and abroad. Along with the development of biotechnology, the microbial preparation with saline-alkali resistance, disease resistance and growth promotion is selected, and the recycling of degraded soil such as moderate and severe saline-alkali soil, continuous cropping obstacle vegetable fields and the like can be realized by improving soil ecology and improving the salt tolerance of plants, so that the method is an economic and effective way.

In nature, due to the selection pressure of ecological environment, such as saline-alkali environment, drought environment and the like, a plurality of plants which are tolerant or adapted to the extreme environment and microorganisms symbiotic with the plants live, and a large number of microorganisms exist in soil, plant roots or plant tissues, wherein the bacterial strains have the functions of preventing diseases, promoting growth or improving the stress resistance of the plants, and have wide application prospects in the aspects of improving the utilization rate of fertilizers, reducing the use of chemical pesticides and the like, so the bacterial strains become hot spots for research, but most of the currently developed and applied bacterial strains have the preference or specificity of plant types or regions, and the application range is limited to a certain extent, so the development cost is increased, and the practical application is inconvenient. Therefore, it is a research and development direction to screen and obtain microbial strains with broad-spectrum characteristics.

The bacillus has strong stress resistance and long survival period, and is distributed in various soils, plant rhizomes and plant tissues. Some strains of the microorganism are used for preparing microbial fertilizers or microbial pesticides due to functions of growth promotion, antagonism and the like, but are mainly applied to conventional soil production, are suitable for medium and severe saline-alkali soil, and particularly have few excellent bacilli suitable for improving various obstacle soils, so that corresponding research and development work needs to be strengthened, and application potential needs to be excavated.

The applicant discloses in an invention patent application with the application number of 201611208244.4, a bacillus amyloliquefaciens ZCM18 separated from saline-alkali soil in Haixing county, the preservation number of the strain is CGMCC No.12959, the strain has the broad-spectrum growth-promoting and stress-resistant effects, the salt resistance is less than or equal to 5%, the strain can be colonized at the rhizosphere of various plants, and substances such as indoleacetic acid, zeatin, polypeptide antibiotics, proline and the like which promote the growth of the plants, inhibit pathogenic fungi and improve the stress resistance and disease resistance of the plants can be secreted.

Disclosure of Invention

The invention aims to provide simple Bacillus (Bacillus simplex) CGMCC No.16458 with high salt tolerance, multifunctional repairing and broad spectrum functions of continuous cropping, saline-alkali and other obstacle soil.

The strain is separated from the rhizosphere of the sow thistle in severe saline-alkali soil (0.68 percent, pH 8.39) in Haixing county. The strain is preserved in the common microorganism center of China general microbiological culture Collection center (CGMCC) in 2018, 9 and 11, the classification name is Bacillus simplex (CGMCC) with the preservation number of CGMCC No.16458, and the address of the preservation organization is located in the microorganism research institute of China academy of sciences (CGMCC) No. 3 of North Xilu No.1 of the Chaozhou south China.

The bacterial colony morphology and physiological and biochemical characteristics of the strain are as follows:

the bacterial colony of the strain is round, white and opaque, smooth and flat, neat in edge, slightly raised in the middle, relatively wet, gram-positive and rod-shaped through gram staining microscopic observation. Has no hemolytic reaction. The strain is identified as the simple bacillus by the morphological characteristics, the physiological and biochemical characteristics and the 16S rRNA sequence analysis of the strain.

The salinity tolerance of the strain is 10 percent.

Under natural conditions, the strain can be colonized at the rhizosphere of various plants, and can dissolve phosphorus, fix nitrogen, produce substances such as siderophin, ACCD, cytokinin and the like, promote plant growth, inhibit pathogenic fungi and improve the stress resistance of the plants.

The culture of the strain: LB medium (5 g yeast powder, 10g tryptone, 5g NaCl5g, 15g agar, 1000mL distilled water) was used.

The method for expanding culture of the strain comprises the following steps:

1. slant culture of strain: inoculating the strain to a testTube slant, culturing at 30-35 deg.C and pH 6.8-7.2 for 2-3 days, shaking with sterile water for 30 min, diluting by 10 times, counting with blood counting plate, and counting bacteria number not less than 10⁹cfu/mL。

2. And (3) shake flask culture: inoculating slant strain into triangular flask containing culture medium according to 0.5-2%, culturing at 30-35 deg.C, pH 6.8-7.2 and rotation speed of 200 rpm for 36-60 hr to make the number of bacteria reach 10⁹cfu/mL。

The culture medium comprises: tryptone 5.0 g; peptone 1.0 g; 2.0g of glucose; k₂HPO₄0.5g；MgSO₄0.5 g; 1000mL of distilled water; the pH value is 6.8-7.2. 15g of agar powder is added into the solid culture medium.

3. Fermentation: placing the sterilized fermentation medium into a 10-30% triangular flask liquid containing amount, and culturing for 24-48 hours under the conditions that the inoculation amount is 1-5% and the temperature is 30-35 ℃; or adding sterilized fermentation medium 40-80% of the volume of the fermentation tank, wherein the inoculation amount of the seed liquid is 2-10%, the temperature is 30-35 deg.C, and the ventilation amount is 0.4-0.6m³The stirring speed is 150-¹⁰cfu/mL。

The fermentation medium comprises the following components: nitrogen source (bean cake powder, tryptone, peptone, beef extract, yeast powder) 5-15 g, carbon source (corn starch, glucose, molasses, bran, rice bran) 5-15 g, MgSO₄1g-5g，KH₂PO₄1g-5g，K₂HPO₄1g to 5g, 1000mL of water, and 6.8 to 7.2 of pH value.

The invention also aims to provide a microbial agent containing simple Bacillus (Bacillus simplex) CGMCC No. 16458.

The invention also aims to provide a biological fertilizer containing simple Bacillus (Bacillus simplex) CGMCC No. 16458.

The fourth purpose of the invention is to provide the application of the simple Bacillus (Bacillus simplex) CGMCC No.16458 and fermentation liquor, microbial agent or biological fertilizer containing the simple Bacillus (Bacillus simplex) CGMCC No.16458 in promoting the growth of plants, wherein the plants are grain crops, economic crops and pasture. The economic crops particularly refer to fruits, vegetables and vegetables.

The fifth purpose of the invention is to provide the application of simple Bacillus (Bacillus simplex) CGMCC No.16458 and fermentation liquor, microbial agent or biological fertilizer containing the simple Bacillus CGMCC No.16458 in preventing and controlling plant diseases, wherein the pathogenic bacteria of the plant diseases comprise pythium aphanidermatum, fusarium, curvularia, microsporum and phytophthora, and the plants comprise grain crops, economic crops and pasture. The economic crops particularly refer to fruits, vegetables and vegetables.

The invention aims to provide the application of simple Bacillus (Bacillus simplex) CGMCC No.16458 and fermentation liquor, microbial agent or biological fertilizer containing the simple Bacillus (Bacillus simplex) CGMCC No.16458 in improving salt tolerance of plants, wherein the salt concentration of the plants in the growth environment is improved by 0.3 percent, and the plants are grain crops, economic crops and pasture grass. The economic crops particularly refer to fruits, vegetables and vegetables.

The improvement of the salt tolerance of the plants means that the salt concentration of the plants in the environment suitable for the growth of the plants is improved by 0.3 percent.

In particular, the germination rate of wheat on moderate saline-alkali land can be recovered to more than 85%.

Specifically, the bacterial suspension of Bacillus simplex CGMCC No.16458 or the fermentation liquor containing the bacterial suspension is sprayed on organic fertilizer particles to prepare the final viable count of 2 × 10⁷-2×10⁹The cfu/g biological fertilizer is used for regulating and controlling the growth of grain crops, pasture and continuous cropping obstacle farmland economic crops in moderate and severe saline-alkali soil.

The invention achieves the substantive characteristics and obvious technical progress that:

the invention adopts a multistage screening method to obtain a strain which can be widely applied to degraded soil such as medium and heavy saline-alkali soil, continuous cropping obstacle vegetable field and the like, simple Bacillus (Bacillus simplex) CGMCC No.16458 of various plants, the salinity resistance of the strain is 10 percent, the strain is proved by the experiment of an applicant to have higher capability of dissolving phosphorus, fixing nitrogen, producing siderophin, ACCD and cytokinin, has stronger antagonism to Pythium, Fusarium, Curvularia lunata, microsporum maydis and Phytophthora (Phytohthora), is suitable for the production of multifunctional biological fertilizers such as disease prevention, growth promotion and saline-alkali resistance, is applied to the production of economic crops such as medium and heavy saline-alkali soil grain crops, pasture, continuous cropping obstacle and the like, promotes the emergence of seedlings, the root development, prevents and controls root diseases, obviously improves the yield of crops and the like, and can reduce the use of chemical fertilizers, has wide application prospect.

Detailed Description

The present invention is further described with reference to the following examples, which are not intended to limit the scope of the present invention, and the claims are not to be interpreted as limiting the scope of the present invention.

Example 1

Morphology and identification of simple Bacillus (Bacillus simplex) CGMCC No.16458

1. Morphological characteristics of the Strain

Round, flat, wet, sticky colonies were formed on LB plates, which grew cloudy in LB liquid medium. By gram staining, the two ends are blunt and round, the length is different, gram-positive bacilli and subterminal spores larger than thalli can be seen under the microscope.

2. Physiological and biochemical characteristics of strain

Experiments prove that the strain can grow on an LB plate with 10 percent of salt content. Experiments show that the strain has the capabilities of dissolving phosphorus, fixing nitrogen and producing siderophin, ACCD and cytokinin. The activity of the ACCD enzyme reaches 7.27 mM/mg/h.

3. Identification and preservation of strains

Through 16S rDNA sequence analysis, comprehensive thallus morphology, physiological and biochemical characteristics and 16S rDNA gene sequence, the strain in the application is Bacillus simplex. The strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms (CGMCC for short, China academy of sciences microorganism research institute No. 3, West Lu 1, Beijing, Toyobo, Naja) in 2016, 9, 11, and the preservation number is CGMCC No.16458 and the classification name is Bacillus simplex (Bacillus simplex).

Example 2

Preparation of biological fertilizer containing simple Bacillus (Bacillus simplex) CGMCC No.16458

Inoculating a simple Bacillus (Bacillus simplex) CGMCC No.16458 bacterial liquid stored in a glycerinum tube to an LB solid culture medium for activation, culturing at 30 ℃ for 2 days, inoculating a single bacterial colony to a seed culture medium, culturing at 30 ℃ for 200 r/min, and carrying out transfer culture once again according to an inoculum size of 1% in mass ratio when the culture is carried out for a late logarithmic phase, wherein the obtained bacterial liquid is the seed liquid. Fermenting with the seed liquid. Adding sterilized fermentation medium 60% of the volume of the fermentation tank, inoculating seed solution 5%, heating to 30 deg.C, and ventilating at 0.5m³Stirring at 200 rpm for 75% dissolved oxygen, defoaming edible soybean oil, and culturing for 45 hr to reach bacteria number of 10¹⁰cfu/mL。

The fermentation medium comprises the following components: 5-15 g of nitrogen source (bean cake powder, tryptone, peptone, beef extract and yeast powder) and 5-15 g of carbon source (corn starch, glucose, molasses, bran and rice bran); MgSO (MgSO)₄1g-5g；KH₂PO₄1g-5g；K₂HPO₄1g-5 g; 1000mL of water; the pH value is 6.8-7.2.

The zymophyte liquid is sprayed on organic fertilizer particles to prepare the organic fertilizer with the content of viable bacteria more than 2 × 10⁷cfu/g of bio-fertilizer.

Example 3

Bacillus simplex (CGMCC No. 16458) test for resisting rot fungi, fusarium, curvularia, microsporum and phytophthora

Inoculating fungus blocks to be tested in the center of an LB plate, symmetrically placing 4 small filter paper sheets with the diameter of 6mm at a position 2.5cm away from the center of the plate, dropwise adding 10 mu L of simple Bacillus (Bacillus simplex) CGMCC No.16458 bacterial liquid on each paper sheet, repeating each treatment for 3 times, and culturing at 30 ℃; as a control, a test fungus block is inoculated at the center of an LB plate, 4 small filter paper sheets with the diameter of 6mm are symmetrically placed at the position 2.5cm away from the center of the plate, 10 mu L of sterile water is dripped on each paper sheet, each treatment is repeated for 3 times, and the culture is carried out at the temperature of 30 ℃. And (5) taking out the plate when the fungi of the control group grow to fill the plate or grow to a paper sheet, measuring the diameter of the fungi, and calculating the inhibition rate.

Inhibition (%) [ 1- (test group fungus diameter/control group fungus diameter) ] x 100

Simple Bacillus (Bacillus simplex) CGMCC No.16458 has certain inhibition effect on pythium, fusarium, curvularia, microsporum and phytophthora (the specific result is shown in Table 1). As can be seen from the table I, the simple Bacillus (Bacillus simplex) CGMCC No.16458 has relatively high inhibition rate on pathogenic fungi, and the simple Bacillus (Bacillus simplex) CGMCC No.16458 has inhibition rates on pythium, fusarium, curvularia, microsporum and phytophthora which are respectively 43.91%, 68.61%, 54.43%, 60.47% and 47.89%.

TABLE 1 inhibition results of Bacillus simplex (Bacillus simplex) CGMCC No.16458 antagonistic pathogenic fungi

Example 4

Test for improving germination rate of wheat seeds in salt environment by simple Bacillus (Bacillus simplex) CGMCC No.16458

Washing thallus centrifugally collected from a simple Bacillus (Bacillus simplex) CGMCC No.16458 culture with sterile water for 3 times, and then suspending the thallus in the sterile water to obtain a simple Bacillus (Bacillus simplex) CGMCC No.16458 bacterial suspension (1 × 10)⁸cfu/mL). Performing surface sterilization on wheat seeds (the germination rate of Jimai 325 is more than or equal to 99 percent), spreading the wheat seeds on a sterile filter paper flat plate, adding 5mL of liquid, wherein the group 1 is sterile water, the group 2 is a bacterial suspension, the group 3 is sterile water containing 0.3 percent of NaCl, the group 4 is a bacterial suspension containing 0.3 percent of NaCl, the group 5 is sterile water containing 0.6 percent of NaCl, the group 6 is a bacterial suspension containing 0.6 percent of NaCl, the group 7 is sterile water containing 0.9 percent of NaCl, the group 8 is a bacterial suspension containing 0.9 percent of NaCl, and the final concentration of the bacterial suspension is 10⁵And counting the germination rate after cfu/mL and dark culture for 2d (the specific result is shown in Table 2).

TABLE 2 Germination Rate statistics Table (Unit:%)

As can be seen from Table 2, Bacillus simplex (CGMCC No. 16458) can increase the germination rate of wheat, and under the environment of 0% NaCl, 0.3% NaCl, 0.6% NaCl and 0.9% NaCl, the germination rates of wheat seeds are respectively increased by 1.15% (P < 0.05), 7.18% (P < 0.01), 11.48% (P < 0.01) and 17.35% (P < 0.01). And the effect is more remarkable as the salt concentration is increased. Under the action of simple Bacillus (Bacillus simplex) CGMCC No.16458 and with the NaCl concentration of 0.3%, the germination rate of wheat is not different from that of 0% NaCl, which shows that the simple Bacillus (Bacillus simplex) CGMCC No.16458 can improve the salt concentration suitable for plant growth by 0.3%, and the simple Bacillus (Bacillus simplex) CGMCC No.16458 can keep the germination rate of wheat in the NaCl environment of 0.9% above 80%, thus having application prospect in the aspect of improving and utilizing saline-alkali soil.

Example 5

Application of fermentation product of Bacillus simplex (CGMCC No. 16458) in Sudan grass in moderate saline-alkali soil

In this example, the bio-fertilizer prepared in example 2 was used. A control biological fertilizer-Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CGMCC No.12959 strain biological fertilizer is prepared by the same method. The field test site is set in Haixing county, Cangzhou city, Hebei province, and the salinity of the soil is 0.4%. The experiment was performed with 4 treatments, repeated 4 times, in a rectangular, fully randomized block design, with a cell area of 10.0 m × 3.0 m to 30.0 m square.

And (3) test treatment:

treatment 1: simple Bacillus (Bacillus simplex) CGMCC No.16458 biological fertilizer and conventional fertilization are reduced by 20 percent

And (3) treatment 2: the weight reduction of the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CGMCC No.12959 biological fertilizer and the conventional fertilizer application is 20 percent

And (3) treatment: conventional fertilization

And (4) treatment: blank control

Wherein the fertilizer to be tested was the one produced in example 2 and containing Bacillus simplex (CGMCC No. 16458) and Bacillus simplex (Bacillus simplex) respectivelyBacillus amyloliquefaciens (Bacillus amyloliquefaciens) CGMCC No.12959 with the bacteria content of 2 × 10⁸cfu/g biological fertilizer (wherein the content of nitrogen, phosphorus and potassium is 5.2%). The conventional fertilization is to apply 40kg of chemical compound fertilizer (nitrogen, phosphorus and potassium content is 42%) per mu, and other field management measures are the same.

The application method comprises the following steps:

treatment 1: simple Bacillus (Bacillus simplex) CGMCC No.16458 biological fertilizer 65 kg/mu + bottom application compound fertilizer (42%, NPK 22:10:10)32 kg/mu;

and (3) treatment 2: bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CGMCC No.12959 biological fertilizer 65 kg/mu + bottom application compound fertilizer (42%, NPK 22:10:10)32 kg/mu;

and (3) treatment: 40 kg/mu of bottom application compound fertilizer (42%, NPK 22:10: 10);

and (4) treatment: no fertilizer is applied.

And (5) mowing 45 days after sowing, and keeping the stubble height at 30 cm above the ground. It can be seen by direct observation before harvesting that Sudan grass in the plot treated 1 grows best, showing that the roots are more thick and the stems are thicker, the average height of grass group is the highest, and the plots treated 1, 2 and 3 grow better than the plot treated 4, the leaves are dark green, and the stems are thicker. Treatment 4 had the smallest leaf, thinnest stem, light green leaf and weakest growth potential (see table 3 for specific results).

TABLE 3 statistics of cell biomass (yield unit: kg)

As can be seen from Table 3, the yield of the Sudan grass in the test field is increased by 83.93 kilograms per mu respectively by applying the simple Bacillus (Bacillus simplex) CGMCC No.16458 bio-organic fertilizer on the basis of 20 percent of the application-reducing compound fertilizer, compared with the conventional fertilizer application, the yield is increased by 16.80 percent, and the yield increasing effect is extremely obvious; compared with the application of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CGMCC No.12959 biological organic fertilizer, the yield is increased by 41.13 kg per mu, the yield is increased by 7.58 percent, and the yield increasing effect is very obvious; compared with the blank control, the yield of the fertilizer is increased by 116.17 kilograms per mu, the yield is increased by 24.85 percent, and the yield increasing effect is very obvious.

Example 6

Test for improving salt tolerance of corn by simple bacillus

The simple Bacillus (Bacillus simplex) CGMCC No.16458 and the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CGMCC No.12959 biological fertilizer prepared by the method in the embodiment 2 take a substrate containing 0.4 percent of NaCl as a culture substrate to carry out a germination test of corns (Zhengdan 958, the germination rate is more than or equal to 98 percent). Each treatment was repeated 4 times for 360 wells, with the treatment settings as follows:

treatment 1: a culture medium;

and (3) treatment 2: the culture medium + Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CGMCC No.12959 biological fertilizer, the proportion is 9: 1;

and (3) treatment: the culture medium + simple Bacillus (Bacillus simplex) CGMCC No.16458 biofertilizer, the proportion is 9: 1 (volume ratio, the same applies below);

and (3) treatment: corn seeds with uniform size are placed in the holes, 1 grain is placed in each hole, and water is quantitatively poured through the holes. Culturing in sunlight greenhouse. And counting the germination rate every other day from 5 days.

The results are shown in Table 4. When the culture medium containing 0.4% NaCl was used as the medium, the control had a slightly late germination time and a germination rate of only about 40%, and treatments 2 and 3 started to sprout on day 6, the final germination rates were 95.8% and 87.9%, respectively (Table 4). Therefore, the simple Bacillus (Bacillus simplex) CGMCC No.16458 biological fertilizer treatment obviously improves the tolerance of the corn plant to the high-salt environment, and is better than the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CGMCC No.12959 treatment.

TABLE 4 comparison of germination (% of different treatments)

	Day 5	Day 6	Day 7	Day 8
					Process 1	0	0	0.2	0.2
Treatment 2	0	85.9	87.8	87.9
					Treatment 3	0	93.0	95.8	95.8

Example 7

Test of simple Bacillus (Bacillus simplex) CGMCC No.16458 fermentation product on 0.2% saline-alkali soil potted corn

Carrying out bacterial liquid fermentation on simple Bacillus (Bacillus simplex) CGMCC No.16458 (treatment 1) and Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CGMCC No.12959 (treatment 2) according to the method of the embodiment 2, and finally obtaining the fermentation liquid with the bacterial number of 1 × 10¹⁰cfu/mL. The matrix adopted by the pot experiment is 0.2% saline-alkali soil. The applicant inoculates the fermentation liquor around wheat seeds (Kenong 2011, germination rate is more than or equal to 98), and each wheat seed is 5 mu L. The control group was inoculated with 5 μ L of sterile water per wheat seed to surround the wheat seed. Repeat 10 times. And (5) carrying out statistics on the plant height, the overground dry weight and the root dry weight of the wheat after 25 days of culture in an illumination room. Final application of Bacillus amyloliquefaciens (Bacillus a)The plant height of the myloliquefasciens CGMCC No.12959 fermentation liquor, the overground dry weight and the root dry weight are respectively increased by 4.45 percent (P is less than 0.05), 14.29 percent (P is less than 0.05) and 16.67 percent (P is less than 0.05) compared with the control group; the plant height of the fermentation liquor of simple Bacillus (Bacillus simplex) CGMCC No.16458 is applied, and the overground dry weight and the root dry weight are respectively increased by 9.33 percent (P is less than 0.01), 21.43 percent (P is less than 0.01) and 25.00 percent (P is less than 0.01) compared with the control group (the specific result is shown in Table 5). The plant height, the overground dry weight and the root dry weight of the two plants are obviously different.

TABLE 5 Bacillus simplex (Bacillus simplex) CGMCC No.16458

Test on 0.2% saline-alkali soil potted wheat

Treatment of	Average plant height (cm)	Average ground dry weight (g)	Average root weight (g)
				Control group	37.50	0.14	0.12
Process 1	41.00	0.17	0.15
				Treatment 2	39.17	0.16	0.14
Treatment 1 increased (%) -over control	9.33％	21.43％	25.00％
				Treatment 2 increased (%) -over control	4.45％	14.29％	16.67％

Example 8

Experiment of biological fertilizer containing simple Bacillus (Bacillus simplex) CGMCC No.16458 in wheat field in saline-alkali soil

By adopting the method of the embodiment 2, the biological fertilizer containing simple Bacillus (Bacillus simplex) CGMCC No.16458 (treatment 1) and Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CGMCC No.12959 (treatment 2) is respectively prepared, and the content of viable bacteria is 1 × 10⁹cfu/g. The field test site is arranged in saline-alkali soil in Haixing county of Cangzhou city in Hebei province, and the salt content of the soil is 0.24 percent. A fertilizing mode: carrying out rotary tillage for 1 time, spreading 18 kg/mu of compound fertilizer (42%), and carrying out rotary tillage for 1 time. A common combined fertilizer and fertilizer sowing machine is adopted, 2 kg/mu of compound fertilizer (42%) is put in a contrast treatment fertilizer groove, sowing is carried out, 16 kg/mu of biological fertilizer (5.2%) is put in a treatment group fertilizer groove, and sowing is carried out. Finally, the dry root weight and the yield of the wheat (rotation selection 103, the germination rate is more than or equal to 82%) are counted. The dry root weight and yield of final treatment 2 were increased by 12.78% and 12.05% over the control group, respectively, with a significant difference; the dry root weight and yield of treatment 1 were increased by 18.52% and 22.83% respectively, which are very different from the control group (see table 6 for specific results). There was no significant difference in dry weight of roots between treatment 2 and treatment 1, and the yield was significantly different.

TABLE 6 application effect of biological fertilizer containing Bacillus simplex CGMCC No.16458 in wheat in saline-alkali soil

Treatment of	Average root weight (g)	Average yield (kg/mu)
			Control	8.53	198.52
Process 1	10.11	243.84
			Treatment 2	9.62	222.45
Treatment 1 increased (%) -over control	18.52	22.83
			Treatment 2 increased (%) -over control	12.78	12.05

Example 9

Pot culture test of biological fertilizer containing simple Bacillus (Bacillus simplex) CGMCC No.16458 on eggplant in continuous cropping obstacle soil

By the method of example 2, raw materials containing Bacillus simplex (CGMCC No.16458 (treatment 1) and Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CGMCC No.12959 (treatment 2) were prepared2 × 10 content of living bacteria⁹cfu/g. Eggplant pot culture test is carried out by using 15-year continuous cropping cucumber greenhouse soil, a blank carrier is used in a control group, a biological fertilizer is used in a treatment 1 or a treatment 2 in a test group, consistent eggplant seedlings are selected, each treatment is carried out for 12 pots, and the process is repeated for 4 times. Statistics were performed after 40 days of incubation in the light room at 25 ℃. Finally, the average leaf number, stem thickness, root dry weight and plant dry weight of ZCM18 are respectively increased by 9.14% (P > 0.05), 33.51% (P < 0.01), 36.17% (P < 0.01) and 41.04% (P < 0.01) compared with the control group; the average leaf number, stem thickness, root dry weight and plant dry weight of P10 were increased by 13.64% (P < 0.05), 42.33% (P < 0.01), 46.81% (P < 0.01) and 48.11% (P < 0.01) respectively, as compared with the control group (see Table 7 for specific results). The average leaf number, stem thickness, and dry weight of the plants differed significantly between treatment 1 and treatment 2.

TABLE 7 test effect of biological fertilizer containing Bacillus simplex CGMCC No.16458 on greenhouse soil potted eggplant of continuous cropping cucumber for 15 years

Treatment of	Average number of blades	Stem diameter (mm)	Root weight (g)	Plant dry weight (g)
					Control	7.33	5.67	0.47	4.24
Process 1	8.33	8.07	0.69	6.28
					Treatment 2	8.00	7.57	0.64	5.98
Treatment 1 increased (%) -over control	13.64	42.33	46.81	48.11
					Treatment 2 increased (%) -over control	9.14	33.51	36.17	41.04

Example 10

Simple bacillus bio-fertilizer field test in continuous cropping obstacle soil cucumber

By adopting the method of the embodiment 2, the biological fertilizer containing simple Bacillus (Bacillus simplex) CGMCC No.16458 (treatment 1) and Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CGMCC No.12959 (treatment 2) is respectively prepared, and the content of viable bacteria is 5 × 10⁸cfu/g. Performing a cucumber field test by using 20-year continuous cropping cucumber greenhouse soil, wherein a spring stubble fertilizing mode comprises the following steps: carrying out rotary tillage for 1 time, spreading the compound fertilizer (42%) 40 kg/mu, carrying out rotary tillage for 1 time, leveling, and ridging, wherein 3500 cucumbers are planted in each mu. And (3) hole-applying blank carriers (160 kg/mu) for a control group, hole-applying biological fertilizers (160 kg/mu) for a treatment group, and selecting consistent cucumber seedlings for planting.Finally, the total yield of the cucumber spring stubble is counted. The cucumber yields of final treatment 2 and treatment 1 were increased by 13.47% (P < 0.01) and 21.35% (P < 0.01), respectively, compared to the control group (Table 8). The cucumber yields of treatment 2 and treatment 1 differed significantly.

TABLE 8 application effect of biological fertilizer containing Bacillus simplex CGMCC No.16458 on greenhouse soil of 20-year continuous cropping cucumber

Treatment of	Yield (kg)
		Control	13885.00
Process 1	16850.00
		Treatment 2	15755.00
Treatment 1 increased (%) -over control	21.35
		Treatment 2 increased (%) -over control	13.47

Claims

1. Bacillus simplexBacillus simplex) The preservation number is CGMCC No. 16458.

2. A Bacillus simplex (Bt) as defined in claim 1Bacillus simplex) CGMCC No. 16458.

3. A Bacillus simplex (Bt) as defined in claim 1Bacillus simplex) A biological fertilizer of CGMCC No. 16458.

4. A Bacillus simplex as defined in claim 1 (C: (C)), (C: (C))Bacillus simplex) Application of CGMCC No.16458 or fermentation liquor containing the same or microbial agent as defined in claim 2 or biological fertilizer as defined in claim 3 in promoting plant growth, wherein the plant is wheat, corn, eggplant, cucumber, sudan grass.

5. A Bacillus simplex as defined in claim 1 (C: (C)), (C: (C))Bacillus simplex) Application of CGMCC No.16458 or fermentation liquor containing the same or microbial agent as claimed in claim 2 or biological fertilizer as claimed in claim 3 in preventing and controlling plant diseases, wherein the pathogenic bacteria of the plant diseases are pythium, fusarium, curvularia, microsporum and phytophthora.

6. A Bacillus simplex as defined in claim 1 (C: (C)), (C: (C))Bacillus simplex) The application of CGMCC No.16458 or fermentation liquor containing the same or the microbial agent of claim 2 or the biological fertilizer of claim 3 in improving the salt tolerance of plants, wherein the salt concentration of the plants in the environment suitable for growth is improved by 0.3 percent, and the plants are wheat.

7. The use as claimed in claim 4, characterized in that it comprises Bacillus simplex (Bt) ((B))Bacillus simplex) The fermentation liquid of CGMCC No.16458 is prepared from Bacillus simplex (Bacillus simplex)Bacillus simplex) CGMCC No.16458 is inoculated into a sterilized culture medium containing a carbon source, a nitrogen source and inorganic salt for culture, wherein the nitrogen source in the culture medium is selected from one or a combination of soybean cake powder, tryptone, peptone, beef extract and yeast powder, and the carbon source is selected from one or a combination of bran, rice bran, corn starch, glucose and molasses.

8. The use according to claim 7, wherein,the method is characterized in that the culture conditions are as follows: the temperature is 30-35 ℃, and the ventilation volume is 0.4-0.6m³The stirring speed is 150-.